The introduction of HAART for treating acquired immunodeficiency syndrome (AIDS) allowed reaching remarkable milestones, turning AIDS into a chronic disease. However, HAART still has several drawbacks, underscoring the demand of new antiretroviral agents with higher genetic barrier. Development of compounds able to inhibit essential but unexplored targets is mostly attractive to reduce selection of drug resistant strains, as reverse transcriptase (RT)-associated ribonuclease H (RH) function. Although RH is a well-validated drug target, no RH inhibitor (RHI) reached clinical approval so far. Since the discovery of structural homologies between integrase (IN) and RH, it was hypothesized that compounds capable to sequester pivotal Mg2+ cofactors, should inhibit both enzymes and, among them, a promising class is represented by diketo acid (DKA) derivatives. However, DKA chain suffers of several pharmacokinetic issues and, therefore, we attempted to overcome these limits by applying a bioisosteric approach. Indeed, starting from our quinolinonyl DKA derivative previously identified as IN inhibitor and recently discovered as potent RHI, we replaced its DKA chain with a carboxylic acid function. The chelation of the two Mg2+ ions should be guaranteed by the combination of such group together with the ketone in 4-position of the quinolinone ring. Thus, in order to define Structure Activity Relationships (SAR) and to identify efficient and specific RHI, we synthesized a new series of quinolinonyl non-DKA derivatives characterized by variously substituted arylalkyl groups in 1-position and small heteroatoms or aromatic rings in position 6 of the quinolinone core, that proved to inhibit RH within the micromolar-submicromolar range.